We have used the homeobox domain, first identified in Drosophila development-regulating genes, to identify genes in the mammalian genome that may be involved in development and differentiation. This proposal will investigate the regulation of expression and function of several members of the Hox-A cluster of homeobox genes and test the hypothesis that these genes function in the development of the male reproductive system and in gametogenesis, using mouse as a model system. We have focused most of our previous efforts on the gene Hoxa-4 because of its abundant and developmentally restricted expression in the male germ line in the adult animal. In the present proposal, we will continue certain novel aspects of the regulation of the expression and function of this gene in the testis and assess the effects of a complete null mutation on gametogenesis. We will also extend our studies to include experiments analyzing the expression and function of members of the Abdominal B class of Hox=A cluster genes, the mutation of one of which (Hoxa-11) has resulted in both male and female sterility. Specifically, we will: [1] Determine the role of Hoxa-4 in testicular function by i) elucidating the molecular basis of a novel Hoxa-4 transcript that is found in mice in which the endogenous Hoxa-4 gene has been mutated by the insertion of a Neo cassette; and ii) generating mice that will completely lack the Hoxa-4 gene, by deletion of the endogenous gene via targeted recombination in ES cells, and examining the effect on gametogenesis and reproductive function; [2] Delineate the regulatory elements specifically responsible for the expression of Hoxa-4 in the germ line, as compared to its embryonic expression, using reporter constructs in transgenic mice; [3] Determine the role of another member of the Hox-A cluster, Hoxa-A cluster, Hoxa-11, in male reproductive system development and function by examining several aspects of the male sterile phenotype that results from targeted mutagenesis of the Hoxa-11 gene; (4) Examine the expression of two other Abd-B class Hox genes which are adjacent to Hoxa-11 in the cluster, namely Hoxa-9 and Hoxa-10, with particular focus on the reproductive system in the embryo and adult. These experiments will include a detailed molecular analysis of their complex transcripts; and [5] Determine the role of Hoxa-9 and Hoxa-10 during embryonic and adult development by generating mutations in these genes by homologous recombination in ES cells. These studies will provide new insight into the function of homeobox-containing genes during mammalian development, and in particular, their potential role in regulating the complex differentiation pathways during development and in the function of the reproductive system.